BD Phosflow T Cell Activation Kit · 23-11749-00 Rev. A 5/2010 Updated sample preparation...

Catalog No. 560750

BD Phosflow™ T Cell Activation KitInstruction Manual

BD Phosflow T Cell Activation Kitii

The information in this guide is subject to change without notice. BD Biosciences reserves the right to change its products and services at any time to incorporate the latest technological developments. Although this guide has been prepared with every precaution to ensure accuracy, BD Biosciences assumes no liability for any errors or omissions, nor for any damages resulting from the application or use of this information. BD Biosciences welcomes customer input on corrections and suggestions for improvement.

TrademarksAlexa Fluor® is a registered trademark of Molecular Probes, Inc. The Alexa Fluor®, Pacific Blue™, and Cascade Blue® dye antibody conjugates in this product are sold under license from Molecular Probes, Inc., for research use only, excluding use in combination with microarrays, or as analyte specific reagents. The Alexa Fluor® dyes (except for Alexa Fluor® 430), Pacific Blue™ dye, and Cascade Blue® dye are covered by pending and issued patents.

Cy™ is a trademark of GE Healthcare. This product is subject to proprietary rights of GE Healthcare and Carnegie Mellon University and is made and sold under license from GE Healthcare. This product is licensed for sale only for research. It is not licensed for any other use. If you require a commercial license to use this product and do not have one, return this material, unopened, to BD Biosciences, 2350 Qume Drive, San Jose, CA 95131, and any money paid for the material will be refunded.

Cytobank and the Cytobank Logo are property of Cytobank, Inc. © 2009 Cytobank, Inc.

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BD, BD Logo and all other trademarks are property of Becton, Dickinson and Company. © 2015 BD

History

Revision Date Change made

23-11749-00 Rev. A 5/2010 Updated sample preparation instructions, added troubleshooting

23-11749-01 Rev. 01 8/2012 Updated for new T-cell antibody cocktail and new compensation antibodies

560750 Rev. 2 3/2015 Updated Warnings section

For Research Use Only. Not for use in diagnostic or therapeutic procedures.

Contents

Chapter 1: About this kit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5Purpose of the kit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6Kit contents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8Storage and safe handling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13

Chapter 2: Before you begin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17Workflow overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18Required materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19

Chapter 3: Preparation of cells and beads . . . . . . . . . . . . . . . . . . . . . 21Treating and staining the cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22Staining BD CompBeads . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27

Chapter 4: Cytometer procedures . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29Running the beads . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30Setting up the workspace for running cells . . . . . . . . . . . . . . . . . . . . 35Running the cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38Data analysis examples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38

Chapter 5: Reference . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45Sample and parameter labels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46Troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51


1About this kit

This section covers the following topics:

Purpose of the kit (page 6)

Kit contents (page 8)

Storage and safe handling (page 13)

BD Phosflow T Cell Activation Kit6


Purpose of the kit

Uses of the kit The BD Phosflow™ T Cell Activation Kit (Catalog No. 560750) provides the reagents necessary to simultaneously analyze multiple T-cell markers with one of several intracellular phosphoproteins for the purpose of studying T-cell subpopulation-specific signaling events. For some studies, cells must be treated with biological response modifiers (eg, polyclonal activators, mitogens, or cytokines, which are not included in the kit) to induce phosphorylated protein expression. The kit enables flow cytometric phosphoprotein analysis of immune cell subsets without prior purification of T cells from fresh whole blood, enabling the analysis of transient phosphorylation events.

Specificantibodies

The kit includes a multicolor cocktail containing fluorescent anti-human CD3, CD4, and CD8 antibodies to identify CD4+ and CD8+ T cells. Single-color Alexa Fluor® 647-conjugated fluorescent monoclonal antibodies specific for the phosphorylated cell-signaling proteins: p38, ERK1/2, Stat1, Stat3, Stat5, and Stat6, are provided as individual reagents and can be used one at a time with the T-cell cocktail for staining. The kit is optimized to evaluate cell-signaling events associated with T-cell activation, including phosphorylation status information on the intracellular MAPK and JAK/STAT signaling pathway responses transduced by human CD4+ and CD8+ T cells.1–4

Other antibodies Single-color, fluorescent CD4 antibodies are provided to label the BD™ CompBead particles that are used as compensation controls.

Chapter 1: About this kit 7


Fluorescencecompensationcontrol beads

Two populations of microparticles are provided.

BD™ CompBead Anti-Mouse Ig, particles, which bind any mouse kappa light-chain-bearing immunoglobulin

BD™ CompBead Negative Control (FBS) particles, which have no binding capacity

When mixed together with a fluorochrome-conjugated mouse Ig, light-chain-containing antibody, the BD CompBeads provide distinct positive and negative (background fluorescence) stained populations that can be used to optimize fluorescence compensation settings manually or by using instrument setup software. The beads allow you to set up for the application and help conserve cells that would otherwise be used to optimize compensation.

Control cells The kit contains two vials each of lyophilized human leucocytes:

BD Phosflow™ Treated Human Control Cells – T Cells: Includes T cells that express upregulated levels of the kit’s targeted phosphoproteins and serve as positive control cells.

BD Phosflow™ Untreated Human Control Cells – T Cells: Includes T cells that express basal levels of the kit's targeted phosphorylated proteins and serve as negative control cells.



The control cells are used as positive and negative controls for staining with fluorescent antibodies specific for the CD markers and phosphorylated cell-signaling proteins. These cells have been fixed and permeabilized and require only immunofluorescent staining after reconstitution and before flow cytometric evaluation. The Treated Human Control Cells - T Cells are treated with multiple activators, so the activation profiles may differ from those obtained from cells treated with a single activator or biological response modifier. Representative data for leucocytes treated with single-response modifiers are presented in Example of single activation profile analysis (page 39).

Buffers The kit contains all buffers necessary for erythrocyte lysis and leucocyte fixation, permeabilization, and immunofluorescent staining. All buffers and monoclonal antibody reagents in this kit have been optimized for their combined use. Do not substitute other reagents.

Kit contents

Reagentinformation

The BD Phosflow T Cell Activation Kit contains the following reagents.



CD marker antibody cocktail

Fluorescent anti-phosphoprotein antibodies

Reagent Details

BD Phosflow™ Human T-Cell (CD4/CD8) Antibody Cocktail

Cocktail component (Clone): Alexa Fluor® 488 anti-Human CD8 (RPA-T8), PE anti-Human CD3 (UCHT1), PerCP-Cy™5.5 anti-Human CD4 (SK3)

Use: To identify CD3+CD4+ and CD3+CD8+ T lymphocytes

Abbreviation: T-Cell Cocktail

Quantity: 1 vial (1 mL)

Amount for staining: 20 µL per sample (for cells derived from 200 µL of human peripheral blood)

Reagent Details

BD Phosflow™ Alexa Fluor® 647 Mouse Anti-ERK1/2 (pT202/pY204)

Clone: 20A

Use: To stain Erk1 phosphorylated at threonine 202 and tyrosine 204 and Erk 2 phosphorylated at threonine 184 and tyrosine 186

Abbreviation: Alexa Fluor® 647 Erk1/2 (pT202/pY204)



BD Phosflow™ Alexa Fluor® 647 Mouse Anti-p38 MAPK (pT180/pY182)

Clone: 36/p38 (pT180/pY182)

Use: To stain p38 MAPK phosphorylated at threonine 180 and tyrosine 182

Abbreviation: Alexa Fluor® 647 p38 MAPK (pT180/pY182)





BD Phosflow™ Alexa Fluor® 647 Mouse Anti-Stat1 (pY701)

Clone: 4a

Use: To stain Stat1 phosphorylated at tyrosine 701

Abbreviation: Alexa Fluor® 647 Stat1 (pY701)




Clone: 4/P-STAT3






Clone: 47






Clone: 18/P-Stat6





Reagent Details



Fluorescent antibodies for compensation

Note: One of the individual Alexa Fluor® 647-conjugated anti-phosphoprotein antibodies can be used to label the BD CompBeads to complete the panel of compensation controls.

Reagent Details

BD Phosflow™ Alexa Fluor® 488 Mouse Anti-Human CD8

Clone: RPA-T8

Use: To stain BD CompBeads for compensation control

Abbreviation: A488 CD8

Quantity: 1 vial (0.2 mL)

Amount for staining: 20 µL per sample

BD Phosflow™ PE Mouse Anti-Human CD4

Clone: RPA-T4


Abbreviation: PE CD4



BD Phosflow™ PerCP-Cy™5.5 Mouse Anti-Human CD3

Clone: UCHT1


Abbreviation: PerCP-Cy5.5 CD3





Fluorescence compensation control beads

Lyophilized human control cells

Reagent Details

BD™ CompBead Anti-Mouse Ig,

Use: To create control beads stained with Alexa Fluor® 488 CD8, PE-CD4, PerCP-Cy5.5 CD3, or Alexa Fluor® 647-phosphoprotein (These beads bind any mouse light-chain-bearing immunoglobulin.)

Abbreviation: Anti-mouse Ig, beads


BD™ CompBead Negative Control

Use: As negative control beads (These beads have no capacity to bind immunoglobulin.)

Abbreviation: Negative beads


Reagent Details

BD Phosflow™ Treated Human Control Cells – T Cells

Use: As a positive staining control cell

Abbreviation: Treated Control Cells

Quantity: 2 vials (1 test per vial)

The cells are treated in such a way that all of the kit's designated phosphorylated target proteins are activated. Each vial contains sufficient cells to stain for a single phosphorylated target protein.

BD Phosflow™ Untreated Human Control Cells – T Cells

Use: As a negative staining control cell

Abbreviation: Untreated Control Cells

Quantity: 2 vials (1 test per vial)

Untreated Control Cells should be stained with the same antibodies as those for the Treated Control Cells.



Buffers

Serum proteins Components in this kit contain a small percentage of serum proteins. The source of all serum proteins is from USDA-inspected abattoirs located in the United States.

Storage and safe handling

Storage Store the fluorescent antibodies, BD CompBeads, Stain Buffer, Treated and Untreated Control Cells, and Lyse/Fix Buffer at 2 to 8°C. Do not freeze. The fluorescent antibodies and BD CompBeads should be protected from prolonged exposure to light.

Store the Perm Buffer III at –20°C.

Reagent Details

BD Phosflow™ Lyse/Fix Buffer (5X)

Use: To lyse erythrocytes and fix leucocytes

Abbreviation: 5X Lyse/Fix Buffer

Quantity: 1 bottle (50 mL)

BD Phosflow™ Perm Buffer III

Use: To permeabilize cells (eg, fixed leucocytes)

Abbreviation: Perm Buffer III


BD Pharmingen™ Stain Buffer (FBS)

Use: To wash and suspend cells for immunofluorescent staining

Abbreviation: Stain Buffer




Warning Researchers are encouraged to review the listed statements prior to use.

Danger

BD Phosflow™ Lyse/FixBuffer (5X) (component 51-9006606) contains 20.35% formaldehyde (w/w), 15.65% diethylene glycol (w/w) and 7.15% methanol (w/w).

Hazard statements

Combustible liquid.

Harmful if swallowed or in contact with skin.

Toxic if inhaled.

Causes skin irritation.

Causes serious eye damage.

May cause an allergic skin reaction.

Suspected of causing genetic defects.

May cause cancer. Route of exposure: Inhalative.

May cause damage to organs. May cause respiratory irritafion.

May cause damage to the kidneys through prolonged or repeated exposure. Route of exposure: Oral.

Precautionary statements

Wear protective gloves / eye protection.

Wear protective clothing.

Do not breathe mist/vapours/spray.

IF ON SKIN (or hair): Remove/Take off immediately all contaminated clothing. Rinse skin with water/shower.



IF IN EYES: Rinse cautiously with water for several minutes. Remove contact lenses, if present and easy to do.

Continue rinsing.

IF INHALED: Remove victim to fresh air and keep at rest in a position comfortable for breathing.

Danger

BD Phosflow™ Perm Buffer III (component 51-9006607) contains 87.68% methanol (w/w).

Hazard statements

Highly flammable liquid and vapor.

Toxic if swallowed, in contact with skin or if inhaled.

Causes damage to the central nervous system. Route of exposure: Oral.

Precautionary statements

Keep away from heat/sparks/open flames/hot surfaces. - No smoking.

Wear protective clothing / eye protection.

Wear protective gloves.

Do nor breathe mist/vapours/spray.

IF ON SKIN (or hair): Remove/Take offimmediately all contaminated clothing. Rinse skin with water/shower.

IF INHALED: Remove victim to fresh air and keep at rest in a position comfortable for breathing.

Some reagents in this kit contain sodium azide. Sodium azide yields highly toxic hydrazoic acid under acidic conditions. Dilute azide compounds in running water before discarding to avoid accumulation of potentially explosive deposits in plumbing.



The lyophilized human control cells contain human blood, serum, or cells, a potential biohazardous material. Use universal precautions when handling. Handle as if the product were capable of transmitting disease. Material used in this product has been tested using FDA-approved methods and found to be negative for Human Immunodeficiency Virus (HIV-1/HIV-2), Hepatitis B Surface Antigen (HBSAG), and antibody to Hepatitis C Virus (HCV). However, no known test method can offer complete assurance that specimens of human origin will not transmit infectious disease. When handling or disposing, follow precautions described in CDC and FDA recommendations and OSHA Bloodborne Pathogen recommendations.


2Before you begin


Workflow overview (page 18)

Required materials (page 19)



Workflow overview

Workflow The overall workflow consists of the following steps:

®

®

Reserve some untreatedcells for staining and analysis.

Suggested sample and parameter labeling strategy is provided to facilitate analysis with Cytobank software.

Reserve some unstained cells.

Chapter 2: Before you begin 19


Required materials

Materials list The following reagents, consumables, and equipment are required for use with the BD Phosflow T Cell Activation Kit:

Fresh whole blood collected with EDTA or heparin anticoagulant

Note: EDTA can potentially affect the actions of some response modifiers.

Appropriate cellular response modifiers, such as cytokines, Phorbol 12-Myristate 13-Acetate (PMA), etc.

Examples:

Distilled or deionized water

1X Phosphate Buffered Saline (PBS)

Falcon® round-bottom 12 × 75-mm polystyrene tubes with caps (Catalog No. 352058), or equivalent

Falcon® 15-mL polypropylene conical tubes (Catalog No. 352097)

Pipets and pipet tips

37°C water bath

Ice bath

Response modifier Vendor Catalog No.

PMA Sigma P-8139

Human IFN- PBL InterferonSource

11101-2

Human IL-2 BD 554603





Vortex mixer

Centrifuge

BD™ LSR II, BD FACSCanto™ II, BD FACSAria™ II, BD LSRFortessa™, or BD FACSCalibur™ flow cytometer, or other flow cytometer equipped with a blue (488-nm) laser, a red (633-nm) laser, and filter configurations appropriate for the detection of Alexa Fluor® 488, PE, PerCP-Cy5.5, and Alexa Fluor® 647 (or allophycocyanin [APC]).

Computer equipped with flow cytometric data acquisition software (eg, BD FACSDiva™, BD CellQuest™ Pro, or BD CellQuest™ software)

Note: For flow cytometric data analysis using Cytobank™ software, your computer must be equipped with a web browser and have access to the internet. Go to bdbiosciences.com/phosflow and click the Cytobank link, or go directly to cytobank.org for more details, including a list of supported web browsers and their requirements.

For proper digital cytometer setup, we recommend using the BD™ Cytometer Setup and Tracking Beads Kit (Catalog No. 642412) and BD FACSDiva software, version 6. See the BD Cytometer Setup and Tracking Beads Kit technical data sheet and the BD FACSDiva Software Reference Manual for instructions.

http://www.cytobank.org

http://www.bdbiosciences.com/phosflow


3Preparation of cells and beads


Treating and staining the cells (page 22)

Staining BD CompBeads (page 27)



Treating and staining the cells

Overview This topic explains how to treat fresh normal human whole blood cells to modify protein phosphorylation. After the treatment, the red blood cells are lysed, and the leucocytes are fixed and permeabilized prior to staining with the cell surface antibody cocktail and one of the phospho-specific antibodies.

Before you begin Ensure that you have all of the necessary materials available. See Required materials (page 19) for details.

Prepare sufficient Lyse/Fix Buffer by diluting 5X Lyse/Fix Buffer to 1X with distilled or deionized water (4 mL of 1X Lyse/Fix Buffer is required for every 200 µL of blood).

Pre-warm the 1X Lyse/Fix Buffer in a 37°C water bath for 15 to 30 minutes.

Prepare response modifier(s) immediately prior to use by diluting in the appropriate diluent(s). Place on ice until use.

Variables thatcan affect results

Treatment: EDTA is the recommended anticoagulant when stimulating with PMA. PMA stimulation of heparinized blood may result in altered cellular light scatter characteristics and greater cell loss.

For response modifiers dissolved in DMSO, keep the final DMSO concentration at or below 0.1% to minimize nonspecific effects. To reduce final DMSO concentration, dilute the stock PMA in PBS immediately prior to use.

Chapter 3: Preparation of cells and beads 23


Lyse/Fix: Prolonged exposure of cells to Lyse/Fix Buffer may result in poor cell surface staining. After the incubation and centrifugation steps, aspirate the supernatant and immediately add 1X PBS to tubes.

Permeabilization: Prolonged exposure to Perm Buffer III at temperatures above 4°C may compromise surface marker staining. To avoid prolonged exposure of the cells to Perm Buffer III, remove buffer immediately after centrifugation and proceed with wash steps.

Staining: Shorter antibody incubation times may result in dimmer staining of some markers. Stain cells for 60 minutes at room temperature.

Procedure Perform all of the procedures in this section in one continuous flow.

Treating cells

1. Label four 15-mL conical tubes:

• Untreated Unstained

• Untreated Stained

• Treated Unstained

• Treated Stained

2. Add 200 µL of blood to each tube.

3. Add the appropriate amount of response modifier (eg, mitogen, cytokine, kinase inhibitor, etc) to the tubes labeled “Treated,” and add an equal volume of buffer to the tubes labeled “Untreated.”

The following table provides human experimental model system information.



Note: The incubation times and temperatures for all examples were 15 minutes at 37°C.

4. Incubate all of the tubes in a 37ºC water bath for the appropriate length of time for your experiment.

5. Lyse erythrocytes and fix leucocytes from treated and untreated blood samples immediately by adding 4 mL of pre-warmed 1X Lyse/Fix Buffer to each of the 15-mL conical tubes.

6. Mix well by inverting 5 to 10 times and incubate the samples in a 37°C water bath for 10 to 12 minutes.

7. Centrifuge at 600g for 6 minutes. Completely remove the supernatant by aspirating without disturbing the cell pellet. The residual volume should be no greater than 50 µL.

8. Quickly vortex each tube to loosen the cell pellet and add 4 mL of 1X PBS to each tube.

9. Centrifuge at 600g for 6 minutes. Completely remove the supernatant by aspirating without disturbing the cell pellet. The residual volume should be no greater than 50 µL.

Responsemodifier Final concentration

Induced targetphosphorylation Reference

PMA 400 nM Erk1/2, p38MAPK (1–3)

hIFN- 40,000 units/mL Stat1 (2)

hIL-2 100 ng/mL Stat5 (2)





Permeabilizing and staining the cells

1. Vortex each tube to loosen the cell pellet. While vortexing each tube, permeabilize the cells by adding 1 mL of cold Perm Buffer III to the tube.

2. Cap the tubes and incubate on ice for 30 minutes.

3. Add 3 mL of Stain Buffer to each tube and immediately centrifuge at 600g for 6 minutes. Completely remove the supernatant by aspirating without disturbing the cell pellet. The residual volume should be no greater than 50 µL.

Note: The cell pellet may appear translucent.

4. Vortex each tube to loosen the cell pellet.

5. Add 2 mL of Stain Buffer to each tube and immediately centrifuge at 600g for 6 minutes. Completely remove the supernatant by aspirating without disturbing the cell pellet. The residual volume should be no greater than 50 µL.

6. Vortex each tube to loosen the cell pellet.

7. Repeat steps 5 and 6.

8. Add 100 µL of Stain Buffer to each tube and mix well. Transfer 100 µL of cell suspension from each sample into an appropriately labeled 12 x 75-mm tube. Keep cells on ice until ready to stain.

Preparing control cells

1. Prepare the lyophilized human control cells.

a. Open the vials containing the lyophilized human control cells (treated and untreated) and reconstitute the cells in 120 µL of Stain Buffer.

b. Gently vortex for 1 to 2 seconds and let stand at room temperature for 5 minutes.



2. Transfer 100 µL of control cells to appropriately labeled 12 x 75-mm tubes. Keep cells on ice until ready to stain.

Staining cell samples and control cells

1. Add the following to the prepared 12 x 75-mm sample and control cells tubes that are being kept on ice.

Determine which phosphorylated target protein you want to stain in the lyophilized control samples.

Note: Unstained cells are used to assess autofluorescence.

2. Vortex all tubes and incubate at room temperature for 1 hour, protected from light.

Note: Compensation beads can be stained during this incubation period. See Staining BD CompBeads (page 27).

3. Wash cells with Stain Buffer by adding 2 mL of Stain Buffer to each tube and immediately centrifuging at 600g for 6 minutes. Aspirate the supernatant.

4. Suspend the stained cells in 300 to 500 µL of Stain Buffer.

Component

Unstained bloodsamples

Stained bloodsamples

Lyophilized controlsamples

Untreatedcells

Treatedcells

Untreatedcells

Treatedcells

Untreatedcells

Treatedcells

Prepared cells 100 µL 100 µL 100 µL 100 µL 100 µL 100 µL

T cell cocktail — — 20 µL 20 µL 20 µL 20 µL

One Alexa Fluor® 647 anti-phospho-protein Ab

— — 20 µL 20 µL 20 µL 20 µL



Next step The samples are ready to be run on the flow cytometer. Acquire samples immediately (optimal) or no longer than 4 hours after preparation. Keep cell samples at 2 to 8°C and protected from light prior to data acquisition.

If you have not already stained the BD CompBeads, proceed to Staining BD CompBeads (page 27).

If you have stained the BD CompBeads, proceed to Cytometer procedures (page 29) for details on setting up the cytometer.

Staining BD CompBeads

Before you begin This step can be performed during the incubation step (step 2) of Staining cell samples and control cells (page 26).

Procedure Staining the beads

1. Set up the Anti-mouse Ig, and Negative beads for fluorescence compensation. Label five 12 × 75-mm polystyrene tubes as follows:

• Negative

• Alexa Fluor® 488

• PE

• PerCP-Cy5.5

• Alexa Fluor® 647



2. Add the following to each tube in the order shown. Vortex the beads thoroughly immediately before dispensing drops.

3. Vortex all tubes and incubate at room temperature for 30 to 60 minutes, protected from light.

4. Wash the beads by adding 2 mL of Stain Buffer to each tube and immediately centrifuging at 600g for 6 minutes. Remove the supernatant by aspirating. The residual volume should be no greater than 50 µL.

Note: Aspirate the BD CompBeads supernatant. Do not decant. Decanting the supernatant of the beads may cause loss of beads.

5. Resuspend the beads in 300 to 500 µL of Stain Buffer. Keep beads at 2 to 8°C, protected from light prior to data acquisition.

6. Use the stained beads to set up compensation prior to acquiring the stained samples.

Next step Proceed to Cytometer procedures (page 29).

Component NegativeAlexaFluor® 488 PE

PerCP-Cy5.5

AlexaFluor® 647

Stain Buffer 100 µL 100 µL 100 µL 100 µL 100 µL

Negative beads 1 drop 1 drop 1 drop 1 drop 1 drop

Anti-mouse Ig, beads — 1 drop 1 drop 1 drop 1 drop

Alexa Fluor® 488 CD8 — 20 µL — — —

PE CD4 — — 20 µL — —

PerCP-Cy5.5 CD3 — — — 20 µL —

Alexa Fluor® 647 anti-phosphoprotein Ab

— — — — 20 µL


4Cytometer procedures


Running the beads (page 30)

Setting up the workspace for running cells (page 35)

Running the cells (page 38)

Data analysis examples (page 38)



Running the beads

Purpose of theprocedure

The stained BD CompBeads are run to calculate and establish fluorescence compensation.

Before you begin The guidelines and examples in this section use a BD FACSCanto II flow cytometer, BD FACSDiva software for acquisition, and the optional Cytobank software for data analysis. However, the fundamental approach to cytometer setup, acquisition, and analysis can be adapted to other flow cytometers and data analysis software applications.

Run the appropriate instrument setup and QC procedures for your flow cytometer. We recommend the use of BD Cytometer Setup and Tracking Beads and BD FACSDiva software, version 6. Ensure that your instrument configuration is appropriate for this assay. See your instrument user’s guide for more information.

Complete the steps in Preparation of cells and beads (page 21).

Chapter 4: Cytometer procedures 31


Procedure To run the prepared compensation control beads:

1. Create a new experiment in BD FACSDiva software.

2. If you have saved application settings for use with this kit, apply the application settings. If no application settings exist, proceed to the next step.

3. In the Cytometer Settings dialog, select the Parameters tab and delete all parameters except FSC, SSC, Alexa Fluor® 488, PE, PerCP-Cy5.5, and Alexa Fluor® 647.

Note: Click the Threshold tab and ensure the threshold value is appropriate to display BD CompBeads.

4. Create compensation controls using the Compensation Setup feature in BD FACSDiva software. Use the generic rather than the antibody-specific labels as indicated in the following figure.



5. Prior to running BD CompBeads, ensure that fluorescence PMT voltages are appropriate for cell analysis.

a. Place a tube of unstained cells on the cytometer and acquire data using the Unstained Control worksheet. Adjust FSC and SSC voltages so that all cell populations are on scale. Record voltages for future use in Running the cells (page 38).

b. Adjust the P1 gate to include the lymphocyte population. Ensure that the fluorescence signals measured from the cells are on scale for all fluorescence parameters. Adjust PMT voltages, if necessary.

c. Place a tube of stained cells, positive for all fluorochromes, on the cytometer and acquire data using the Unstained Control worksheet. Ensure that the fluorescence signals measured from the cells are on scale for all fluorescence parameters. Adjust PMT voltages, if necessary.



6. Save the application settings for future use.

a. In the Browser, right-click Cytometer Settings and select Application Settings > Save.

b. Name the application, then click OK.

7. Place the tube of Negative beads on the cytometer and acquire data using the Unstained Control worksheet. Adjust FSC and SSC voltages such that the bead population is on scale.

Note: BD CompBeads require higher FSC voltage settings than cells.

8. Set the P1 gate around the singlet bead population.



9. Record data for Negative beads and each of the stained BD CompBeads. Make sure to adjust the P2 histogram regions to fit the positive populations.

10. Calculate compensation.

a. From the Experiment menu, select Compensation Setup > Calculate Compensation.

b. Name the compensation setup, then click Link and Save.

Note: Do not adjust the fluorescence settings. Adjusting the settings now will invalidate the compensation calculations.

Next step Proceed to Setting up the workspace for running cells (page 35).

Additionalresources

See Getting Started with BD FACSDiva Software for information about creating and working with experiments.

See the BD FACSDiva Software Reference Manual for information about creating compensation controls, creating statistics views, acquiring data, and calculating compensation.



See the BD Cytometer Setup and Tracking Application Guide for information about applying application settings.

Setting up the workspace for running cells

Before you begin Complete the steps in Running the beads (page 30).

If you have previously saved a template for use with this kit, import the template and proceed directly to Running the cells (page 38).

Procedure To set up the workspace for running cells:

1. Create tubes and label them appropriately as shown below.

2. Enter the detector voltages for FSC and SSC from step 5 in Running the beads.

3. Toggle to the Global Worksheet and create the plots shown in the following figure.



4. Place a tube of cells stained with the T-cell cocktail and Alexa Fluor® 647 anti-phosphoprotein on the cytometer. Acquire sufficient cells to create the gates shown in the preceding global worksheet figure. Label the gates as shown.

5. On the Labels tab of the Experiment Layout window, enter parameter labels for each marker in the experiment as shown in the following figure.

Note: For more complex experiments, see Sample and parameter labels (page 46).



6. Save this worksheet as a template for use in future experiments.

Next step Proceed to Running the cells (page 38).

Additionalresources

See Getting Started with BD FACSDiva Software for information about working in the BD FACSDiva workspace.

See the BD FACSDiva Software Reference Manual for information about how to import analysis templates.



Running the cells

Before you begin Complete the steps in Staining BD CompBeads (page 27) and Setting up the workspace for running cells (page 35).

Procedure To run the cells:

1. Apply a Stopping Gate to the T-cell population using the Acquisition Dashboard.

2. Acquire a sufficient number of events to ensure statistically meaningful data of all subpopulations being studied.

3. On the Dashboard, ensure that Storage Gate is set to record All Events.

4. Record data for all tubes.

5. You can use BD FACSDiva software for the analysis, or you can export FCS data files in the appropriate format for your third-party analysis software, such as Cytobank software.

Note: Cytobank software can open both FCS 2.0 and FCS 3.0 data files.

Data analysis examples

About this data The experiments shown in these examples were prepared and acquired as outlined in this manual, then analyzed using Cytobank software.



Cytobanksoftware

Cytobank flow cytometry analysis software may be used to analyze your T-cell signaling data. Cytobank facilitates the calculation of phosphoprotein fold-change values and allows you to visualize your data in a variety of ways, such as dot plots, heatmaps, and histogram overlays.

Analysis using Cytobank requires access to the internet and a recommended web browser. Detailed instructions on uploading data and analyzing it using Cytobank software, as well as the appropriate browser recommendation can be found by clicking the Cytobank link at bdbiosciences.com/phosflow, or going directly to cytobank.org/manual.

Example ofsingle activationprofile analysis

The following example shows activated CD4+ and CD8+ T-cell signaling profiles for each phosphoprotein marker identified by the BD Phosflow T Cell Activation Kit. The sample was whole blood from a healthy human donor.

Note: Phosphoprotein staining may vary depending on the cell sample source and treatment.

Gating Strategy

The first plot shows a light scatter gate used to identify peripheral blood cell populations. Next, a CD3+ lymphocyte population is identified within the Intact Cell gate. Finally, CD8+ and CD4+ T-cell populations are identified within the CD3+ population.

All Cells

All Cells

Intact Cells

Intact Cells CD3+ Cells

CD3+ Cells

CD4+ Cells

CD8+ Cells

Cell Populations

Forward Scatter CD3-PE CD4-PerCP-Cy™5.5

Sid

e S

ca

tte

r

Sid

e S

ca

tte

r

CD8-

Alex

a Fluo

r® 48

8

96.6%

37.3%CD3+

CD8+ T cellsIntactCells

31.9%15.9% CD4+ T cells

http://www.cytobank.org/manual

http://www.bdbiosciences.com/phosflow



Activation profiles

The following figure shows the activation profiles for CD4+ and CD8+ T cells. The data are displayed as histogram overlays using Cytobank software. These histograms show the signaling response of each phosphorylation marker induced by the corresponding response modifier listed in the following table.

Phosphorylation marker Response modifier

Erk1/2 (pT202/pY204) p38MAPK (pT180/pY182)

PMA

Stat1 (pY701) Human IFN-Stat3 (pY705) Human IL-6

Stat5 (pY694) Human IL-2

Stat6 (pY641) Human IL-4

CD8+ Cell Activation Profiles

CD4+ Cell Activation Profiles

p-ERK p-p38 p-STAT1 p-STAT3 p-STAT5 p-STAT6

Fold Change

1.0 7.0 13.0

Control

PMA

IL-2

IL-4

IL-6

Control

PMA

IL-2

IL-4

IL-6

p-ERK p-p38 p-STAT1 p-STAT3 p-STAT5 p-STAT6



Example of adose-responseanalysis

The following example shows a p-Stat5 signaling response to various doses of IL-2 in CD4+ and CD8+ T cells. The sample was whole blood from two healthy human donors. Data were collected using a BD FACSCanto II flow cytometer and analyzed and displayed in a variety of ways using Cytobank software.

Note: The fluorescence intensity frequency distributions of phosphoprotein-positive and -negative cell populations in a sample may vary depending on the cell sample source and treatment.

Gating strategy

The gating strategy used is the same as shown in Example of single activation profile analysis (page 39).

Dose-response analysis

The following series of plots shows the dose-response analysis of CD3+ cells. The contour plots depict a comparison of CD8 and p-Stat5 staining to show that CD8– and CD8+ T cells have a differential sensitivity to IL-2.

CD3+ T Cells, Titration of IL-2

0

CD8 Alexa Fluor® 488

p-S

TA

T5-A

lexa F

luor®

647

100 ng/ml 50 ng/ml 25 ng/ml 12.5 ng/ml 6.25 ng/ml

3.12 ng/ml 1.56 ng/ml 0.78 ng/ml 0.39 ng/ml 0.2 ng/ml 0.1 ng/ml

0.59% 0.26%

63.19% 35.97%

57.35% 36.51%

4.9% 1.24%

54.99% 34.11%

7.34% 3.56%

45.59% 23.25%

16.86% 14.30%

32.58% 10.61%

30.17% 26.63%

24.03% 4.32%

38.70% 32.96%

21.02% 2.84%

41.80% 34.35%

17.70% 1.82%

44.80% 35.68%

13.46% 1.68%

48.85% 36.01%

9.48% 1.11%

52.98% 36.43%

7.01% 0.75%

53.91% 38.32%

5.28% 0.80%

56.76% 37.15%



The following histogram overlays show the response of p-Stat5 to varying doses of IL-2 in CD4+ and CD8+ T cells for one donor.

CD4+ T cells

Titration of IL-2

CD8+ T cells

0 ng/mL

100 ng/mL

50 ng/mL

25 ng/mL

12.5 ng/mL

6.25 ng/mL

3.12 ng/mL

1.56 ng/mL

0.78 ng/mL

0.39 ng/mL

0.20 ng/mL

0.10 ng/mL

p-STAT5-Alexa Fluor® 647

Fold Change

1.0 9.5 18.0



The following heatmap, displaying CD4+ and CD8+ T-cell data, shows the increase in p-Stat5 response to varying doses of IL-2, as represented by the change in color. In Cytobank, moving the cursor over any square in the heatmap allows you to see the underlying data.

The fold-change values of the p-Stat5 response to IL-2 in CD4+ and CD8+ T cells can be exported to a third-party spreadsheet program for further analysis. In the following figure, exported fold-change values for donor 1 are plotted against varying doses of IL-2 to determine other metrics such as EC50 values.

Titration of IL-2

Fold Change

1.0 9.5 18.0

0 ng

/mL

100

ng/m

L

50 n

g/m

L

25 n

g/m

L

12.5

ng/

mL

6.25

ng/

mL

3.12

ng/

mL

1.56

ng/

mL

0.78

ng/

mL

0.39

ng/

mL

0.20

ng/

mL

0.10

ng/

mL

CD4+ T cellsCD8+ T cells

p-STAT5CD

4

p-STAT5-Alexa Fluor® 647

p-S

TA

T5 (fo

ld c

hange)

Titration of IL-2

0

2

4

6

8

10

12

14

16

18

IL-2 (ng/ml)

CD4+ T cells

CD8+ T cells

0.1 1 10 100




5Reference


Sample and parameter labels (page 46)

Troubleshooting (page 48)

References (page 51)



Sample and parameter labels

Before you begin We recommend identifying individual parameters, specimens, and tubes with unique, descriptive labels. This will expedite data analysis using Cytobank software.

Parameter labels Label each fluorescence parameter with the name of the antibody specificity.

Note: The phospho-protein should be specifically identified by the parameter label (for example, p-ERK-Alexa Fluor 647, not p-protein-Alexa Fluor 647).

Specimen andtube labels

Label each specimen and tube with text indicating how it is unique within the experiment. Labels should be at least three characters long and should include enough experimental information to uniquely identify each tube. Include one or more of the following:

Condition. Stimuli and inhibitors represent different conditions (eg, IL-2, IL-6, PMA, inhibitor14, drug35). Growth media and states of cells (eg, fresh cells, frozen cells) also represent conditions.

Timepoint. Timepoints are specified durations used in the experiment. Most commonly, these are times that samples were allowed to signal before being fixed (eg, 30 s, 5 min, 4 h).

Dose. Dosage titrations are various specified concentrations used in the experiment (eg, 0.1 ng/mL, 10 ng/mL, or 5 µM).

Individual. Individuals can be different donors or animals (eg, donor3, BALB/c5).

Chapter 5: Reference 47


Sample (specimen) type. Types can include major classifications of the samples tested (eg, wild type vs knock out, tumor vs normal, cell lines vs whole blood, spleen, or lymph node).

A good example of a tube label:

01 healthy whole blood donor1 IL-2 10 ng-mL 15 min

Note: Use the Specimen and Tube Name fields if using BD FACSDiva software or the Sample Name field if using BD CellQuest Pro software.

Compensationand controllabels

Label samples used for compensation and controls with text indicating how the sample is unique within the experiment. For example:

Compensation samples. Include “comp” in the sample name (eg, comp_PE).

Lyophilized control cells. Include “lyo” in the name of all controls derived from lyophilized human cells (eg, lyocell control).

Other controls. If other controls or beads are used, include the word “control” or “bead” in the sample name.

Number Sample type Individual Condition Dose Timepoint

01 healthy whole blood

donor1 IL-2 10 ng/mL 15 min



Troubleshooting

Recommendedaction

These are the actions we recommend taking if you encounter the following problems.

Problem Possible cause Recommended action

No or incomplete red blood cell lysis

Lyse/Fix buffer diluted in 1X PBS

Use water to dilute the 5X Lyse/Fix Buffer to 1X.

Inadequate mixing with blood

Immediately after adding the Lyse/Fix Buffer to blood samples, mix them thoroughly by inverting the tubes at least 5 to 10 times.

Inadequate incubation time

After mixing the Lyse/Fix Buffer with blood samples, incubate them for 10 to 12 minutes at 37°C.

Improper whole blood to Lyse/Fix ratio

Use 20 times the volume of whole blood for the Lyse/Fix step.



No or poor surface CD marker staining

Cells are exposed to Lyse/Fix Buffer for too long

Incubation time in the Lyse/Fix Buffer should not exceed 15 minutes at 37°C.

Delay in centrifugation After the Lyse/Fix step, the Lyse/Fix Buffer should be removed immediately by centrifuging the cells, removing the supernatant, and washing the cells once with 1X PBS.

Cells are exposed to Perm Buffer III for too long at temperatures greater than 4°C

Do not exceed the recommended permeabilization time—30 minutes on ice.

After the permeabilization step, immediately remove the Perm Buffer III by centrifuging the cells, removing the supernatant, and washing the cells with the Stain Buffer.

Inadequate washing After permeabilization, immediately add the recommended amount of Stain Buffer to the tubes, centrifuge, and follow with an additional two washes.

Residual volume too large after Lyse/Fix and permeabilization steps

Residual volume should be no greater than 50 µL after each wash.




No or poor activation

No biological response modifier added

Add the appropriate amount of biological response modifier.

Biological response modifier too old, expired, or lost activity

Prepare fresh biological response modifier and store as aliquots. Use new aliquots for all experiments. Avoid repeated freeze/thaw cycles.

No carrier protein in the dilution buffer for cytokines

Use a buffer (such as PBS) containing at least 1 mg/mL of BSA to dilute cytokines, and keep the diluted stimulant on ice until use.

Diluted biological response modifier kept too long before use

We recommend using stimulants within 30 minutes of dilution.

Inappropriate biological response modifier selected

As a positive control, use the suggested biological response modifier at the recommended concentration, incubation time, and temperature.




References

Cited references 1. Perez OD, Mitchell D, Campos R, Gao GJ, Li L, Nolan GP. Multiparameter analysis of intracellular phosphoepitopes in immunophenotyped cell populations by flow cytometry. Curr Protoc Cytom. 2005;Chapter 6:Unit 6.20.

2. Montag DT, Lotze MT. Successful simultaneous measurement of cell membrane and cytokine induced phosphorylation pathways [CIPP] in human peripheral blood mononuclear cells. J Immunol Methods. 2006;313:48–60.

3. Montag DT, Lotze MT. Rapid flow cytometric measurement of cytokine-induced phosphorylation pathways [CIPP] in human peripheral blood leukocytes. Clin Immunol. 2006;121:215–226.

4. Perez OD, Mitchell D, Jager GC, et al. Leukocyte functional antigen 1 lowers T cell activation thresholds and signaling through cytohesin-1 and Jun-activating binding protein 1. Nat Immunol. 2003;4:1083–1092.



Notes



Notes



Notes

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BD Phosflow T Cell Activation Kit · 23-11749-00 Rev. A 5/2010 Updated sample preparation...

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